1. Field of the Invention
The present invention relates to improvements in total internal reflection (TIR) spectroscopy, and more particularly to an apparatus and method for TIR-based spectroscopic imaging with improved angular scanning, particularly attenuated total reflection (ATR) imaging spectroscopy and microscopy.
2. Description of the Related Art
Attenuated total reflection (ATR) spectroscopy, also known as internal reflection spectroscopy (IRS), originates in the observation by Newton almost two centuries ago that a propagating wave of radiation which undergoes total internal reflection (TIR) in a higher index of refraction medium in contact with a lower index of refraction medium gives rise to an evanescent field in the lower refractive index medium. It was, however, not until 1960 that this phenomenon was exploited for producing absorption spectra. Since then, numerous ATR-based applications have been developed, many of them in the biosensor field. In biosensors based on ATR, the evanescent field probes a thin layer (penetration depth about one wavelength) of sample-containing material at a solid/liquid interface, resulting in a measurable attenuation of the reflected radiation in proportion to the imaginary part of the refractive index of the thin layer, i.e., absorption spectroscopy.
As is well-known, the spectrum in ATR-spectroscopy consists of a reflectance vs. wavelength curve or spectrum, where the angle of incidence may be scanned in order to vary evanescent wave penetration depth (see e.g., Harrick, N. J., Internal Reflection Spectroscopy, Harrick Scientific Corp., New York, 1967; and Mirabella, F. M. Jr., Internal Reflection Spectroscopy: Review and Supplement, Harrick Scientific Corp., New York, 1985). Photon absorbance at the ATR-sensor surface, with or without evanescent electrical field strength enhancing metal film or particles, is detected as a more or less sharp and deep minimum, or dip, in the internal reflectance (TIR) curve, and the depth (absorbance peak) is a measure of the amount of detected sample, and the wavelength is used to identify the kind of molecule.
A variant of ATR-spectroscopy, hereinafter referred to as SPR-spectroscopy, is based on surface plasmon resonance (SPR) in an electrical field-strengthening layer of metal film or metal islands or particles applied on the solid medium at the solid/liquid interface. The angle (or wavelength) corresponding to the minimum or centroid of the photon absorbance peak, or reflectance dip, and/or the change in angle, is a measure of the amount of detected sample. More particularly, the change in SPR-angle and/or SPR-wavelength is a measure of the change in the real part of the refractive index (n) and/or the change in the thickness (d) of the sample interaction layer or layers at the solid/liquid interface. If the sample also allows photon absorbance, this can be measured via changes in the shape (reflectance minimum and dip width) of the SPR-ATR-curve.
The surface concentration of the sample can be calculated from the shift in SPR-angle and/or SPR-wavelength by use of well-known relations between surface concentration and layer structural parameters n, d, and empirical or calculated refractive index increment of the sample solute (De Feijter, J. A., et al., Biopolymers 17:1759, (1978); and Salaman, Z., et al., Biochemistry 33:13706, (1994)).
In some SPR-spectroscopic optic configurations, e.g., using both p- and s-polarized light, the photon absorbance is converted into a peak rather than a dip in the reflectance curve, and the angle (or wavelength) position, or shift in position, for this peak is used as a quantitative measure of the amount, or change in amount, of detected sample.
SPR-based sensors are commercially available for use in research and development, for example the BIACORE® instrument line from Biacore AB, Uppsala, Sweden. These instruments use a sensor glass chip covered with a thin gold film and an integrated fluid cartridge for passing sample fluid and other fluids over the sensor chip. A fan shaped beam of light is coupled to the sensor chip via a prism such that an angular range of incident light is reflected internally along a line at the glass/gold film interface creating a plasmon evanescent field at the gold film/fluid interface, and the reflected light intensity distribution versus angle of incidence for a row of sensor spots along the illuminated line, is detected by a photodetector array.
The sensitivity in the detectable change of the angle (or wavelength) at the dip or peak (or in some cases, dips or peaks) of the SPR spectrum is mainly limited by the degree of constancy, drift and noise of the background light intensity of the TIR-curve. Ideally, the TIR-curve is constant versus angle. In practice, however, due to variation of reflectance with the angle of incidence, and the radiation distribution from the light source, the TIR-curve will generally be a gaussian type curving line with at least one maximum and which at its low and high angle ends, respectively, is more or less sloping. The sensitivity of SPR-spectroscopy is, of course, particularly in the case of kinetic studies (measurement of the time dependence of the reflectance curve, shape and/or position), higher the more stable the light intensity at total internal reflection is over the angular or wavelength range of interest, i.e., the less dependent the “background” TIR-curve is on the angle (or wavelength).
The sensitivity of SPR-spectroscopy can thus be further increased if the TIR-curve is normalized, such as by a computer software algorithm. This usually requires highly stable TIR-curve data, i.e., low temporal noise and drift, and that the TIR-curve has as little curvature and sloping as possible and is as smooth as possible. Such computerized normalization of the TIR-curve is, for example, done in the BIACORE® instruments mentioned above, where the normalization procedure is performed on the TIR-spectrum with a sample of refractive index high enough to give TIR for the angle range used. Due to the use of a focused light beam (spot or line) and a static optical system in the present BIACORE® instruments, the illuminated sensor surface for TIR is fixed (stationary). The stability of the provided irradiance is therefore only limited by noise and drift in the light source.
Whereas conventional ATR-spectroscopy measures the surface characteristics of a sample at a fixed small spot or a thin line, spatial scanning of the incident light beam across a surface area may produce an image of the spectral property distribution over the scanned area. This technique is usually referred to as ATR-microscopy. Thus, for instance, various arrangements for surface plasmon microscopy (SPM) have been proposed for detection of the spatial distribution of the refractive index. Rather than spatially scanning a focused line or light beam across the sensor surface area with time, it has also been described to momentarily illuminate and image the whole surface area in question and scan the incident light angle or wavelength with time. A biosensor based on ATR-microscopy may, for example, be used for analyzing a large number, or an array, of sample spots simultaneously.
An example of an ATR-sensor apparatus of the last-mentioned type based on scanning the angle of incidence of a probing collimated (parallel) beam which illuminates the whole surface area is disclosed in WO 98/34098 (the full disclosure of which is incorporated by reference herein). A representative illustration of this system is given in FIG. 1 herein, where a light source, LS, illuminates a collimator optics, CO, to produce a parallel light beam. The beam passes an interference filter, I, as a monochromatic beam and impinges on a first flat scanner mirror, SM1, to be deflected onto a second scanning mirror, SM2. The latter deflects the beam into a prism Pr for coupling the light into a sensor surface, SS, the beam being totally internally reflected at the sensor interface side of the coupling prism. The p-polarized component of the beam then passes a polarizer, P, whereupon the beam is directed into a spherical objective, SO, to produce a real image on a photo detector array or matrix, D, of the sensor surface area from its reflected light intensity pattern.
The detector array D is arranged such that the real image of the sensor area is produced on a rectangular part, D, of the array, the objective SO having its real image plane positioned at the plane of the photodetector array. Numerals 1, 2 and 3 at the detector array D denote the respective images of the corresponding subzones on the sensor surface SS, indicated at 1′, 2′ and 3′, respectively.
The two scanning mirrors SM1 and SM2 have a related rotational or oscillating movement to produce an angularly scanned collimated beam incident within a range of angles of incidence on the sensor surface side of the prism Pr. “Beam walking” of the illuminated area, which is caused by refraction of the collimated light at the entrance of the prism Pr during the scan of the angle of entrance and would give an irregular irradiance (radiant power per unit area) at the sensor surface, is avoided by synchronizing the movements of the scanning mirrors SM1 and SM2 (as well as adapting the distances and angles between mirrors and prism, and scanned angular range of the mirrors) to provide a fixed center for the intersection of the incident collimated light beam with the sensor surface SS.
In the general type of ATR-microscope described above, the momentary incident angle of the collimated light beam irradiating the sensor surface during the angle scan may de determined by various means. WO 98/34098 proposes to determine the momentary incident angle by detecting a part of the light beam reflected at the sensor surface on a second detector. More particularly, the objective SO comprises an additional part having its back focal plane positioned at the plane of the photodetector array to permit a minor part of the reflected collimated beam to be focused onto a minor, linear part of the detector area so that each angle of reflection corresponds to a specific linear detector position within the detector area.
While in the ATR-microscope described in WO 98/34098 the fixed center of the illuminated area thus gives a more stable irradiance, or optical power distribution across the cross-section of the beam at the sensor surface than in the case of beam walking, this ATR-microscope has another shortcoming.
Thus, in WO 98/34098, as in conventional ATR-spectroscopy, the center angle of the collimated beam is orthogonally incident onto the entrance surface of the ATR-prism. During the angle scan of this collimated beam, however, the angle of incidence at the entrance surface of the prism will be oblique, a typical angle range being, say, 15°. Therefore, even for an illuminated ATR-sensor area with eliminated beam walking, i.e., a fixed center during angle scan, there will be a variation in the light intensity, or irradiance, with the angle of incidence since the intersection area of the incident collimated beam (of mainly constant beam cross-section area) with the ATR-surface will vary in accordance to Lambert's Cosine Law. The length of the intersection area thus has a fixed center but the length may be said to “oscillate” around its center.
For example, if the length of the intersection area is denoted L, the length L=(beam diameter)/cosine(angle of incidence), or, in case of a rectangular beam cross section, L=(beam cross-section length)/cosine(angle of incidence). This causes, even if the beam walking has been eliminated, L (and the illuminated area) to increase, and the irradiance to decrease, the larger the angle of incidence at the sensor area. The irradiance is proportional to cosine(angle of incidence), during the angle scan by the following exemplary factors: for angle of incidence scan 63° to 77°: 2.0×; for angle of incidence scan 62° to 82°: 3.37×; and for angle of incidence scan 64° to 84°: 4.19×.
This monotone continuously decreasing light intensity with cosine(angle of incidence) creates a correspondingly sloping TIR-data vs. angle curve. As is readily seen, the reflectance at 77° will be about 50% of that at 63°, and at 84° only about 25% of that at 63°.
Such a substantial decrease in light intensity with increased angle of incidence can not readily be compensated for by computerized data processing like the above-mentioned normalization of the TIR-curve, or at least not without a complex normalization method and software. There is therefore a need for means that overcome the problem of varying light intensity during angular scan in ATR-based microscopy.
Related to ATR-microscopy are other microscopic techniques also based on the evanescent wave phenomenon at total internal reflection without or with evanescent field enhancing metal films or textures, such as, for example, total internal reflection fluorescence (TIRF), total internal reflection phosphorescence, and scattered total internal reflectance (STIR). To overcome the above-mentioned problem of varying light intensity during angular scan would therefore be valuable also in those as well as in other related TIR-techniques.